Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro

AbstractThe embryonic origin and development of hematopoietic and endothelial cells is highly interdependent. We hypothesized that primary endothelial cells from murine yolk sac and para-aortic splanchnopleura (P-Sp) may possess the capacity to expand hematopoietic stem cells (HSCs) and progenitor cells ex vivo. Using Tie2-GFP transgenic mice in combination with fluorochrome-conjugated monoclonal antibodies to vascular endothelial growth factor receptor-2 (Flk1) and CD41, we have successfully isolated pure populations of primary endothelial cells from 9.5-days after coitus (dpc) yolk sac and P-Sp. Adult murine bone marrow Sca-1+c-Kit+lin- cells were cocultured with yolk sac or P-Sp Tie2-GFP+Flk-1+CD41- endothelial cell monolayers for 7 days and the total number of nonadherent cells increased 47- and 295-fold, respectively, and hematopoietic progenitor counts increased 9.4- and 11.4-fold, respectively. Both the yolk sac and P-Sp endothelial cell cocultures facilitated long-term (> 6 months) HSC competitive repopulating ability (2.8- to 9.8-fold increases, respectively). These data suggest that 9.5-dpc yolk sac- and P-Sp-derived primary Tie2-GFP+Flk-1+CD41- endothelial cells possess the capacity to expand adult bone marrow hematopoietic progenitor cell and HSC repopulating ability ex vivo. (Blood. 2003;102:4345-4353)

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Cell-nonautonomous function of Id1 in the hematopoietic progenitor cell niche

Blood ◽

10.1182/blood-2008-09-179788 ◽

2009 ◽

Vol 114 (6) ◽

pp. 1186-1195 ◽

Cited By ~ 20

Author(s):

Hyung Chan Suh ◽

Ming Ji ◽

John Gooya ◽

Michael Lee ◽

Kimberly D. Klarmann ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cell ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Cytokine Levels ◽

Cell Niche ◽

Marrow Cellularity

Abstract Development of hematopoietic stem cells (HSCs) and their immediate progeny is maintained by the interaction with cells in the microenvironment. We found that hematopoiesis was dysregulated in Id1−/− mice. Although the frequency of HSCs in Id1−/− bone marrow was increased, their total numbers remained unchanged as the result of decreased bone marrow cellularity. In addition, the ability of Id1−/− HSCs to self-renew was normal, suggesting Id1 does not affect HSC function. Id1−/− progenitors showed increased cycling in vivo but not in vitro, suggesting cell nonautonomous mechanisms for the increased cycling. Id1−/− HSCs developed normally when transplanted into Id1+/+ mice, whereas the development of Id1+/+ HSCs was impaired in Id1−/− recipients undergoing transplantation and reproduced the hematologic features of Id1−/− mice, indicating that the Id1−/− microenvironment cannot support normal hematopoietic development. Id1−/− stromal cells showed altered production of cytokines in vitro, and cytokine levels were deregulated in vivo, which could account for the Id1−/− hematopoietic phenotypes. Thus, Id1 is required for regulating the hematopoietic progenitor cell niche but is dispensable for maintaining HSCs.

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Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽

10.1182/blood.v104.11.1686.1686 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1686-1686

Author(s):

Hideyuki Oguro ◽

Atsushi Iwama ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Target Genes ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem ◽

Ex Vivo Culture ◽

Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

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Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽

10.1182/blood.v110.11.1677.1677 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1677-1677

Author(s):

Zejin Sun ◽

Yanzhu Yang ◽

Yan Li ◽

Daisy Zeng ◽

Jingling Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Ex Vivo ◽

Bone Marrow Failure ◽

Wild Type ◽

Hematopoietic Stem ◽

Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

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Hematopoietic progenitor/stem cells immortalized byLhx2 generate functional hematopoietic cells in vivo

Blood ◽

10.1182/blood.v99.11.3939 ◽

2002 ◽

Vol 99 (11) ◽

pp. 3939-3946 ◽

Cited By ~ 43

Author(s):

Perpétua Pinto do Ó ◽

Karin Richter ◽

Leif Carlsson

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Cell Lines ◽

Embryonic Stem ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Generate Functional ◽

Hematopoietic Stem

Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whetherLhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor–dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell–deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell–deficient hosts.

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Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Nature Communications ◽

10.1038/s41467-021-27263-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christina M. Termini ◽

Amara Pang ◽

Tiancheng Fang ◽

Martina Roos ◽

Vivian Y. Chang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Endothelial Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem ◽

Vascular Regeneration ◽

Vascular Niche ◽

Hematopoietic Reconstitution ◽

Neuropilin 1

AbstractIonizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

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Role of microvascular endothelial cells on proliferation, migration and adhesion of hematopoietic stem cells

Bioscience Reports ◽

10.1042/bsr20192104 ◽

2020 ◽

Vol 40 (3) ◽

Author(s):

Fanli Lin ◽

Shuyue Wang ◽

Hao Xiong ◽

Yang Liu ◽

Xiaoming Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Microvascular Endothelial Cells ◽

Cell Contact ◽

Hematopoietic Stem ◽

Direct Cell ◽

Cell Cell

Abstract Background: The present study investigated the effects of microvascular endothelial cells (MECs) on the chemotaxis, adhesion and proliferation of bone marrow hematopoietic stem cells (HSCs) ex vivo. Methods and Results: MECs were collected from the lung tissue of C57BL/6 mice, and HSCs were isolated with immunomagnetic beads from bone marrow of GFP mice. MECs and HSCs were co-cultured with or without having direct cell–cell contact in Transwell device for the measurement of chemotaxis and adhesion of MECs to HSCs. Experimental results indicate that the penetration rate of HSCs from the Transwell upper chamber to lower chamber in ‘co-culture’ group was significantly higher than that of ‘HSC single culture’ group. Also, the HSCs in co-culture group were all adherent at 24 h, and the co-culture group with direct cell–cell contact had highest proliferation rate. The HSC number was positively correlated with vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) levels in supernatants of the culture. Conclusions: Our study reports that MECs enhance the chemotaxis, adhesion and proliferation of HSCs, which might be related to cytokines SDF-1 and VEGF secreted by MECs, and thus MECs enhance the HSC proliferation through cell–cell contact. The present study revealed the effect of MECs on HSCs, and provided a basis and direction for effective expansion of HSCs ex vivo.

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Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽

10.1182/blood.v104.11.1688.1688 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1688-1688 ◽

Cited By ~ 1

Author(s):

Noriko Miyake ◽

Ann C.M. Brun ◽

Mattias Magnusson ◽

David T. Scadden ◽

Stefan Karlsson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

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In Vitro and In Vivo Selection of Genetically Modified Cells Using shRNA Against HPRT and Treatment with 6-thioguanine.

Blood ◽

10.1182/blood.v110.11.2590.2590 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2590-2590

Author(s):

Christopher C. Porter ◽

James DeGregori

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Genetically Modified ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Stem ◽

Selection Of

Abstract Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy trials. Thus, the ability to select for or amplify a population of cells that has been modified to express a gene of interest might enhance the effectiveness of gene therapy. Strategies for in vivo expansion of genetically modified cells that have been studied to date have relatively high toxicity or low efficacy in selection of hematopoietic stem cells. We hypothesized that resistance to the purine analog 6-thioguanine (6TG) could be programmed via lentiviruses, and that treatment with 6TG would allow for selection of genetically modified cells in vitro and in vivo. Using short hairpin RNAs, we achieved efficient knockdown of hypoxanthine phosphoribosyl transferase (HPRTkd), the enzyme required for 6TG cytotoxicity, in the murine hematopoietic progenitor cell line FL5.12. In so doing we were able to provide Fl5.12 cells with resistance to 6TG. In the presence of 6TG, HPRTkd cells continued to proliferate for at least 30 days, whereas control transduced cells ceased proliferating after 7-10 days. 6TG treatment of mixed cultures of GFP+-HPRTkd cells and untransduced cells resulted in selective outgrowth of HPRTkd cells. Knockdown of HPRT in FL5.12 cells was found to attenuate the checkpoint activation, cell cycle arrest and apoptosis seen in control transduced cells when treated with 6TG. Knockdown of HPRT in murine primary hematopoietic cells also allowed for selection of transduced cells with 6TG ex vivo. Furthermore, and most importantly, after transduction of whole bone marrow and transplantation into sub-lethally irradiated recipient mice, a single, short course of treatment with 6TG resulted in up to 12 fold greater percentages of circulating transduced granulocytes as compared to untreated controls. These results suggest that genetically modified hematopoietic stem cells can be selected in vivo using 6TG. This strategy may be useful for therapy of a variety of hematopoietic diseases, particularly those that affect hematopoietic progenitors. The benefits of this strategy include the following: 1) the use of a lentivirus with a self inactivating long terminal repeat, 2) a very short cassette encoding drug resistance, making the vector easier to manipulate, and 3) a very well tolerated and relatively non-toxic medication for selection.

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Cord Blood Hematopoietic Progenitor Cell in Co-Culture with Bone Marrow Mesenchymal Stromal Cells: A Synergic Effect

Blood ◽

10.1182/blood.v118.21.4811.4811 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4811-4811

Author(s):

Camillo Almici ◽

Arabella Neva ◽

Rosanna Verardi ◽

Simona Braga ◽

Andrea Di Palma ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Progenitor Cell ◽

Cord Blood Sample ◽

Hematopoietic Stem

Abstract Abstract 4811 The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures: No relevant conflicts of interest to declare.

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Novel Method to Study Mouse Bone Marrow Endothelial Cells in Vivo and in Vitro

Blood ◽

10.1182/blood.v120.21.617.617 ◽

2012 ◽

Vol 120 (21) ◽

pp. 617-617 ◽

Cited By ~ 2

Author(s):

Yuxin Feng ◽

Ming Liu ◽

Fukun Guo ◽

Wei Liu ◽

Leesa Sampson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Culture System ◽

Assay Method ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Egfp Reporter

Abstract Abstract 617 Self-renewal, differentiation, and proliferation of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) are maintained in a complex microenvironment of the adult bone marrow (BM). BM endothelial cells (ECs) have been proposed to be a key component of HSC and LSC niche. However, in contrast to the well-developed culture system of human ECs, current work of murine BM endothelial cells is hindered by a lack of mouse bone marrow endothelial cell primary culture and suitable assay methods to clearly define murine BMEC functionality in vivo and in vitro, which limits genetic and mechanistic studies by using mouse models. To establish an in vivo approach to study the EC function in adult mice, a strain of Tie2-CreER transgenic mice was generated to allow conditional and inducible manipulation of BMECs by Cre recombinase expression under the Tie2 promoter. In vivo lineage tracing was achieved in a Tie2-CreER/TD-tomato or -EGFP reporter mouse strain. Upon a four day Tamoxifen injection regimen, TD-tomato or EGFP reporter was readily visualized in bone marrow vasculature that colocalizes with CD31+ ECs as determined by immunostaining. FACS analysis of Tie2-CreER/EGFP reporter mice showed that the EGFP+ cells in the BM were exclusively in the CD45- VEGFR2+ and CD31+ cell fraction, with no EGFP+ cells being detectable in the CD45+ hematopoietic lineages or osteoblast/stroma cell fractions, suggesting that the Tie2-driven CreER expression is limited to the endothelial lineage in the adult BM. Next, we developed an in vitro method to culture and assay the mouse BMECs functionally. An in vitro selection process allowed us to establish a primary BM cell culture condition that permitted functional expansion and maintenance of mouse BMECs in long-term tissue culture, yielding homogenous CD45- cells expressing endothelial markers CD31, CD34 and VEGFR2. These cells formed capillary-like structures in 2-demensional and 3-demensional tubes/capillaries, and showed TD-tomato reporter color when derived from the Tamoxifen induced Tie2-CreER/TD-tomato mouse BM. They showed expected adhesion and migration activities and morphology of ECs. Lineage chasing assays using isolated CD45+ and CD45- BM cells from the Tie2-CreER/Td-tomato mice demonstrated that the BMECs in our culture system, bearing the Tie2-promoter driven TD-tomato color and CD31+ marker, were exclusively derived from CD45- non-hematopoietic lineage. Taken together, we have established a faithful assay method for studying murine BM EC functions in vivo and in vitro, allowing the tracking and genetic manipulation of adult BM ECs in mice and in culture. The method can be useful for delineating molecular and cellular mechanisms of BMEC regulation and EC-mediated BM niche function, and may have value in testing anti-angiogenic activities of anticancer drugs in animal models. Disclosures: No relevant conflicts of interest to declare.

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